Substances relating to testing of blood-coagulation



United States Patent O SUBSTANCES RELATING TO TESTING OF BLOOD-COAGULATION Roy E. Speck, 109 Brunswick Drive, Greenwood, Ind. 46142 No Drawing. Filed Mar. 15, 1965, Ser. No. 439,941 Int. Cl. C12k 1/10; G01u 33/16 US. Cl. 19599 13 Claims ABSTRACT OF THE DISCLOSURE A partial thromboplastin time test reagent and a multifactor coagulation reagent, having a chemical activator, the reagents providing good stability, high sensitivity, and a more reproducible and accurate evaluation of a patients coagulability state. Hageman factor is maximally activated in a constant manner, yielding precision and reliability of test results. The multi-factor coagulation reagent is sensitive to both the intrinsic and extrinsic coagulation systems, advantageously permitting both systems to be tested in a single diagnostic procedure.

This invention relates to a new discovery in medical diagnosis, specifically in the field of laboratory diagnosis of coagulation disorders, and more particularly, in substances and methods useful therein and relating thereto.

CURRENT BLOOD COAGULATION THEORY-INTRINSIC TYPE For purpose of clarification and background, a brief description of the current coagulation theory is presented introductorily, as now set forth.

If blood from a persons vein is placed in a glass testtube and observed, it is seen, after a certain length of time, that the blood changes from fluid to a gel. This is the process of coagulation of the so-called intrinsic type. It is termed intrinsic for the reason that all of the coagulation factors involved in the reaction are of intravascular origin. As explained below, the process occurs, basically, in a three-step reaction: (1) formation of blood thromboplastin, (2) conversion of rothrombin, (3) production of gel (clot).

CURRENT THEORYFORMATION OF BLOOD THOMBOPLASTIN Platelets (blood cells of a certain type) release one or more platelet factors as a result of physical contact with the surface of the glass test tube. The platelet factor reacts with several coagulation factors present in normal blood, comprising anti-hemophilia globulin, plasma thromboplastic antecedent, plasma thromboplastic component, Stuart-Prower factor, Hageman factor (which is also activated by the glass surface), calcium ions, and perhaps other unknown factors to produce blood thromboplastin.

Diagrammatically, this might be shown as follows:

Platelet factor, :plus

Plasma thromboplastic antecedent, plus Plasma thromboplastic component, plus Stuart-Prower factor, plus Hageman factor (surface activation), plus Antihemophilia globulin, plus Calcium ions Blood thromboplastin 3,486,981 Patented Dec. 30, 1969 CURRENT THEORYCONVERSION OF PROTHROMBIN Blood thromboplastin, in the presence of other factors, comprising Stuart-Prower factor, proaccelerin, and calcium ions, converts prothrombin to thrombin.

Prothrombin Blood thromboplastin, plus Stuart-Prower factor, plus Proaccelerin, plus Calcium ions Thrombin CURRENT THEORY-PRODUCTION OF GEL (CLOT) Thrombin reacts with the soluble plasma protein, fibrinogen, and converts it to the insoluble fibrin in the form of a gel.

Fibrinogen Thrombin Fibrin INTRINSIC COAGULATION-TIME AND OTHE FACTORS This intrinsic system is a relatively slow reaction. The time required for the reaction to go to completion depends on several conditions, such as temperature, concentration of coagulation factors, and degree of physical surface activation.

CURRENT BLOOD COAGULATION THEORY--EXTRINSIC TYPE CURRENT THEORY-CONVERSION OF PROTHROMBIN Tissue thromboplastin in the presence of Stuart-Prower factor, proaccelerin, proconvertin, and calcium ions, converts prothrombin to thrombin.

Prothrombin Tissue thromboplastin, plus Proconvertin, plus Proaccelerin, plus Stuart-Prower factor, plus Calcium ions Thrombin CURRENT THEORYPRODUCTION OF GEL (CLOT) Thrombin reacts with fibrinogen, and converts it to the insoluble fibrin.

Fi'brino gen Thrombin Fibrin 3 EXTRINSIC COAGULATION-TIME AND OTHER FACTORS, AND COMPARISON TO INTRINSIC SYSTEM The extrinsic system of coagulation is relatively rapid (10-15 seconds), in contrast to the intrinsic system of coagulation (4-5 minutes). There are some other basic differences. Blood thromboplastin does not require proconvertin for its activity in conversion of prothrombin to thrombin, whereas, tissue thromboplastin does. Surface activation has a dramatic effect on the intrinsic system, but only a minor one on the extrinsic system.

CHEMICAL ACTIVATION OF HAGEMAN FACTOR The new discovery mentioned in the first paragraph involves, as one thing, the chemical activation of Hageman factor. This process, to be described below, makes it possible to improve certain laboratory tests for the assessing of the activity of certain coagulation factors of blood. This chemical activation of Hageman factor includes the following procedures and components:

(1) Use of ellagic acid, or one of its related compounds or derivatives of these compounds, in the partial thromboplastin time test;

(2) Process for preparing a stable solution of the chemical activator;

(3) Preparation of a reagent consisting of a platelet substitute and the chemical activator; and its use as a blood test for testing coagulation factors; and

(4) Preparation of a reagent consisting of a platelet substitute, chemical activator, and tissue thromboplastin; and its use as a blood test for testing coagulation factors.

THE USE OF ELLAGIC ACID, OR ONE OF ITS RELATED COMPOUNDS OR ONE OF THE DERIVATIVES OF THESE COMPOUNDS, IN THE PARTIAL THROMBOPLASTIN TIME TEST 'PTT TESTCOAGULATION DEFICIENCIES The partial thromboplastin time test (PTT) is a laboratory test used to screen for coagulation disorders.

It detects deficiencies in the following coagulation factors:

It does not detect deficiencies of the following coagulation factors:

(1) Platelets;

(2) Proconvertin;

(3) Calcium; or

(4) Fibrin stabilizing factor.

Hageman factor;

Stnart-Prower factor;

Plasma thromboplastic antecedent; Plasma thromboplastic component; Anti-hemophilia globulin; Prothrombin;

Proaccelerin; and

Fibrinogen.

The test will also detect the presence of circulatmg anti-coagulants, such as anti-thromboplastin and antithrombin,

4 PT TESTGENERAL NATURE The PTT test is an in vitro simulation of the intrinsic system of coagulation. The basic reagent is a platelet substitute in optimum concentration. A platelet substitute is used because of the technical difficulty of preparing a standard platelet suspension.

PTT TESTORIGINAL VERSION The PTT test of Langdell, Wagner, and Brinhous (J. Lab. and Clin. Med., vol. 41, page 637, 1953) is performed in the following manner: The platelet substitute is mixed with oxalated or citrated plasma. To this is added suflicient calcium in solution to neutralize the anti-coagulant and to provide an optimal concentration of calcium ions for the coagulation reaction. The test is performed at 37 C. The time required for the formation of a clot is determined. In case of an abnormality, the time is prolonged. The normal range for the original procedure is 40 to seconds.

PTT TEST-DEFECTS OR DIFFICULTIES OF ORIGINAL VERSION The main defect of the above procedure lies in the fact that glass is an unreliable physical activator of Hageman factor. It neither activates maximally nor consistently. Even the manner in which glass is washed influences the degree to which it will activate Hageman factor. Reproducibility is thus poor.

PTT TESTS-ASUBSEQUENT MODIFICATIONS Attempts have been made to remedy this unreliability by utilizing insoluble activators, such as kaolin, celite, bentonite, and others. These activators do activate maximally, but the cencentration of the activator is critical. If there is not enough activator, maximal activation does not occur. If too much activator is used, absorption of Hageman factor, and perhaps other factors, occurs, resulting in loss of activity.

Furthermore, the time of maximum activity is critical and of short duration, so that the test must be performed at an accurately timed interval of activation.

The normal range for the procedure utilizing koalin or celite is 35 to 45 seconds. This version of the PTT test increased the sensitivity of the test, mainly due to the increased reproducibility of results. However, there are still disadvantages, as mentioned above, particularly with respect to time of activation and concentration of the activator.

PTT TESTCHEMICAL ACTIVATION These disadvantages have been overcome according to the present invention, by the introduction of a chemical activator of Hageman factor. Using a solution of ellagic acid, activation was rapid, less than one minute. Also, the activation was stable. The plasma remains fully activated for as long as 60 minutes. Ellagic acid was used with three commercial partial thromboplastins in separate tests: Cephaloplastin (Dade), Platelin (Warner-Chilcott), and Thrombofax (Ortho). Equivalent results were obtained with each. The normal range was 32. to 38 seconds. The standard deviation was 0.5 second, and represents superior precision. The short normal range represents increased sensitivity to coagulation defects.

PREPARATION OF STABLE SOLUTION OF CHEMICAL ACTIVATOR Ellagic acid was reagent prepared according to Ratnoff and Crum (J. of Lab. and Clin. Med., vol. 63, pages 359377, 1964), but it was not stable, and it precipitated after standing a few days. This was remedied according to the present invention by converting the ellagic acid to its sodium salt, by adding sodium hydroxide, this reaction, it is believed, being as follows:

HO OH 4NaOH :1

Ellagic Acid Sodium Ellagate This was followed by filtration. The resulting solution retained the full activity of the ellagic acid.

COMBINING PARTIAL THROMBOPLASTIN AND A SALT OF ELLAGIC ACID IN A SINGLE REAGENT Utilizing the previous principles, the sodium salt of ellagic acid and partial thromboplastin may be prepared in a single reagent; and each component is found to have no deleterious elfect on the other over an extended time. This reagent functions identically as do the components when used separately.

COMBINING PARTIAL THROMBOPLASTIN, OF A SALT ELLAGIC ACID, AND TISSUE THROMBO PLASTIN IN A SINGLE REAGENT In order to make a reagent sensitive to both the intrinsic and extrinsic systems, tissue thromboplastin was incorporated in the above reagent, at a concentration set forth and discussed hereinbelow.

PREPARATION AND TEST DETAILS (I) Preparation of stable ellagic acid solution (sodium salt) (H) Preparation of stable ellagic acid solution (potassium salt) (1) 0.034 gm. mole) ellagic acid is weighed on analytical balance and transferred to a 50 ml. beaker.

(2) 5 ml. 0.1 M potassium hydroxide are added to dissolve the ellagic acid. (Suflicient potassium hydroxide is added to convert the ellagic acid to potassium ellagate with a slight excess of potassium hydroxide remaining.)

(3) Dilute to 1000 ml. with distilled water in a 1000 m1. volumetric flask, stopper, mix well.

(4) Filter through a 5.0, pore millipore filter.

(III) Preparation of stable ellagic acid solution (ammonium salt) (1) 0.034 gm. (10 mole) ellagic acid is weighed on analytical balance and transferred to a 50 ml. beaker.

(2) 5 ml. 0.1 M ammonium hydroxide are added to dissolve the ellagic acid. (Sufficient ammonium hydroxide is added to convert the ellagic acide to ammonium ellagate with a slight excess of ammonium hydroxide remaining.)

(3) Dilute to 1000 ml. with distilled water in a 1000 ml. volumetric flask, stopper, and mix well.

(4) Filter through a 5.0 pore millipore filter.

(IV) Comparison of ellagic acid and its salts in the partial thromboplastin time test Dilutions were made With distilled water of the sodium ellagate, potassium ellagate, and ammonium ellagate solutions, so that the following concentrations were obtained: 10 10 10- 10-*, molar.

The above dilutions of each of the ellagate solutions (as well as ellagic acid) were tested in the partial thromboplastin test using pooled normal plasma (5 blood donors), a patient under dicou'marol therapy (acquired deficiencies of Prothrombin, proconvertin, Stuart-Prower factor), one patient with hemophilia (deficiency of antihemophilia globulin), and one patient with Christmas disease (deficiency of plasma thromboplastic component).

The results are listed in Charts I, II, III, and IV.

Concentration of Ellagic Acid (sec) 10- M l0- M 10- M 10- M 10- M Pooled Normal Plasma 34 33 34 34 32 Hemophiliac Plasma 128 130 128 129 130 Christmas Disease Plasma 114 115 116 115 Dicoumarolized Plasma 118 118 120 118 CHART II.PARTIAL THROMBOPLASTIN TIME USING SODIUM ELLAGAGE AT VARIOUS CONCENTRATIONS IN COMBINATION WITH PLATELET SUBSTITUTE PRE- PARED ACCORDING TO THE PROCEDURE OF BELL AND ALTON Concentration of Sodium Ellagate (sea) 10- M 10- M 10- M 10 M 10 M Pooled Normal Plasma 36 33 36 38 37 Hemophiliac Plasma 126 126 125 Christmas Disease Plasma. 110 114 114 112 110 Dicoumarolized Plasma 116 118 118 120 118 CHART III.PARTIAL THROMBOPLASTIN TIME USING POTASSIUM ELLAGATE AT VARIOUS CONCENTRATIONS IN COMBINATION WITH PLATELET SUBSTITUTE PRE- PARED ACCORDING TO THE PROCEDURE OF BELL AND ALTON Concentration of Potassium Ellagate (see.)

10- M 10- M 10- M 10- M 10 M Pooled Normal Plasma. 33 36 33 32 33 Hemophiliac Plasma 132 131 131 128 127 Christmas Disease Plasma. 116 118 114 116 118 Dicoumarolized Plasma 120 119 116 118 120 CHART IV.PARTIAL THROMBOPLASTIN TIME USING AMMONIUM ELLAGATE AT VARIOUS DILUTIONS IN COMBINATION WITH PLATELET SUBSTITUTE PRE- PARED ACCORDING TO THE PROCEDURE OF BELL AND ALTON Concentration of Ammonium Ellagate 10- M 10' M 10- M 10- M 10- M Pooled Normal Plasma 37 38 35 37 34 Hemophiliac Plasma 130 134 131 130 129 Christmas Disease Plasm 115 118 116 118 117 Dlcoumarolized Plasma 120 118 118 117 118 (V) Procedures used for tests (1) A commercial partial thromboplastin reagent, Cephaloplastin (Dade) was reconstituted with the various dilutions of the ellagate solutions.

(2) The blood specimens used were collected in 0.1 volume 0.1 M sodium citrate. The blood was centrifuged at 1500 rpm. for 10 minutes, and the supernatant plasma removed. All of the plasmas were tested within thirty minutes of the collection.

(3) 0.1 ml. reconstituted Cephaloplastin was placed in two 10 x 75 mm. glass test tubes.

(4) To each test tube was added 0.1 ml. plasma to be tested, and the test tubes were placed in a 37 C. water bath for three minutes in order for the mixture to come to 37 C.

(5) 0.1 ml. 0.02 M calcium chloride was blown into the mixture, and simultaneously a stop watch was started. After thirty seconds, the tube was gently tilted, and the time required for formation of clot was noted.

(VI) Results (VII) Various hydroxy-substituted aromatic compounds in the partial thrombolastin time test molar solutions of the sodium salts of the following hydroxy-substituted compounds: 3,4-dihydroxyphenylethylamine (dopamine), 1,2 dihydroxy anthraquinone (alizarin), 2,7,8,9 -tetrahydroxytropone (purpurogallin) and 4,4,5,5',6,6'-hexahydroxydiphenic acid, 2,6:2,6"-dilactone (ellagic acid), were prepared in the same manner as in section I, and used in the same manner as in section V, above.

(VIII) Results Equivalent results were obtained with all solutions, showing that these hydroxy-substituted compounds are interchangeable in the partial thromboplastin time test. See Chart V, below.

CHART V.PARTIAL THROMBOPLASTIN TIME USING VARIOUS HYDROXY-SUBSTITUTED AROMATIC COM- POUNDS IN COMBINATION WITH PLATELET SUBSTI- TUTE PREPARED ACCORDING TO PROCEDURE OF BELL AND ALTON. THE CHEMICAL ACTIVATORS WERE USED AT A CONCENTRATION OF 10- M Seconds Sodium Sodium" Sodium Purpuro Sodium Dopamine Alizarin gallin Ellagate Pooled Normal Plasma 36 35 36 34 Hcmophiliac Plasma 133 128 131 128 Christmas Disease Plasma. 117 119 118 117 Dicoumarolized Plasma- 121 116 119 116 *Aged for 48 hours. Aging necessary for maximum activity.

(IX) Comparison of various platelet substitutes combined with sodium ellagate in the partial thromboplastin time test CHART VI.PARTIAL THROMBOPLASTIN TIME USING SODIUM ELLAGATE (10 M) AND VAROUS PLATELET SUBSTITUTES Type of Platelet Substitute (sec) Cephalo- Plateliu plastin Thrombofax (Warner- (Dede) (Ortho) Chilcott) Pooled Normal Plasma 34 37 40 Hemophiliac Plasma 124 128 125 Christmas Disease Plasma Dicoumarolized Plasma (X) Effect of various temperatures on the degree of activation in the partial thromboplastin time test, using platelet substitute and sodium ellagate CHART VII.EFFECT OF TEMPERATURE ON DEGREE OF ACTIVATION IN THE PARTIAL THROMBOPLASTIN TIME TEST USING SODIUM ELLAGATE (10- M) AND i111$PLASTIN (DADE). ACTIVATION TIME, FIVE (XI) Stability of activation in the partial thromboplastin time test, using platelet substitute and sodium ellagate CHART VIIL-STABILITY OF ACTIVATION IN THE PARTIAL THROMBOPLASTIN TIME USING SODIUM ELLAGATE (10- M) AND CEPHALOPLASTIN (DADE). ACTIVATION TEMPERATURE, 37 C.

Activation time in minutes Pooled Normal Plasma (sec) 34 34 34 35 34 Hemophillac Plasma (sec.) 126 128 125 125 126 Christmas Disease Plasma (sec.) 116 116 117 116 116 Dicoumarolized Plasma (see) 110 112 110 113 110 (XII) Multi-factor coagulation reagent (A) Partial thromboplastin time reagent utilizing platelet substitute and chemical activator may be made sensitive to both the intrinsic and extrinsic coagulation systems by incorporation of the appropriate amount of tissue thromboplastin into the partial thromboplastin reagent.

This sensitivity to all factors (except platelets) of both intrinsic and extrinsic coagulation systems is a sensitivity far greater than merely a sensitivity to plasma thromboplastic component, Stewart-Prower factor, proconvertin, and prothrombin, as in the Thrombotest of Owren (Saertrykk av Farmakoterapi nr. 4, vol. XV, 1959. Utgiver: Nyegaard & Co. A/S, Oslo).

The amount of tissue thromboplastin used is that quantity that gives clotting times intermediate between the one-stage Prothrombin Time (Quick, A.J.: The Hemorrhagic Diseases and the Physiology of Hemostasis, p. 312. Charles C. Thomas, Springfield, Ill., 1942) and the Partial thromboplastin time, a desired mixture being in the range of 1% to 16% as shown in Chart IX.

CHART IX.-EFFECT OF VARIOUS CONCENTRATIONS OF TISSUE THROMBOPLASTIN WITH OPTIMUM CONCENTRATION OF SODIUM ELLGATE (10 M) AND CEPHALOPLASTIN (DADE) IN A MODIFIED PARTIAL 'IJIJISIIIJtIOLIBOPLASTIN TIME TEST. NORMAL PLASMA MODIFIED PARTIAL THROMBOPLASTIN TIME IN SECONDS PERCENT TISSUE THROMBOPLASTIN (B) Each lot of the reagent must be standardized by preparation of a dilution curve such as shown in Chart IX. The reagent is stabilized either by incorporating 3% phenol or by freeze drying.

Accordingly, it is seen that the concepts of the present invention provide reagents which do the following:

(1) Makes available a more sensitive partial thromboplastin time reagent.

(2) Makes available a PTT reagent in which Hageman factor is maximally activated and this activation remains constant throughout the test procedure.

(3) Makes available a PTT reagent which is stable with with storage.

(4) Makes available a multi-factor reagent sensitive to both the intrinsic and extrinsic coagulation system.

(5) Makes available a multi-factor reagent stable with storage.

What is claimed is:

1. A partial thromboplastin time test reagent comprising a platelet substitute, and ellagic acidas the chemical activator.

2. A partial thromboplastin time test reagent comprising a platelet substitute, and sodium ellagate as the chemical activator.

3. A partial thromboplastin time test reagent comprising a platelet substitute, and potassium ellagate as the chemical activator.

4. A partial thromboplastin time test reagent comprising a platelet substitute, and ammonium ellagate as the chemical activator.

5. A partial thromboplastin time test reagent comprising a platelet substitute, and a monovalent salt of ellagic acid as the chemical activator.

6. A partial thromboplastin time test reagent comprising a platelet substitute, and an hydroxy-substituted aromatic compound as the chemical activator.

7. A partial thromboplastin time test reagent comprising a platelet substitute, and a salt of an hydroxy-substituted aromatic compound as the chemical activator.

8, A partial thromboplastin time test reagent compris- 7 ing a platelet substitute, and a monovalent salt of an hydroxy-substituted aromatic compound as the chemical activator.

2 w i l References Cited UNITED STATES PATENTS 4/1965 Owren 167-84.5

OTHER REFERENCES Ratnolf et al.: J. Lab. and Clin. Med. 63, #3, March 1964, pp. 359477.

Langdell et al.: J. Lab. and Clin. Med., April 1963, pp. 637-647.

Jurd, L.: J. Am. Chem. Soc., 78, 3445-8 (1956); Chem. Abstr. 51, 1107a (1957).

Hyland Reference Manual of Coagulation Procedures, 1964, pp. 15-18.

Proctor et al.: Amer. J. of Clin. Path. 36, #3, pp. 212- 219, September 1961.

MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,486,981 December 30, 1969 Roy E. Speck It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

Column 2, line 25, "OTHE" should read OTHER Column 4,

line 71, "was reagent" should read reagent was Column 5, lines 40 and 41, "OF A SALT" should read A SALT OF Column 6, line 5, "acide" should read acid line 30, "PRL-" should read PRE- line 42, "ELLAGAGE" should read ELLAGATE Signed and sealed this 10th day of November 1970.

(SEAL) Attest:

WILLIAM E. SCHUYLER, JR.

Edward M. Fletcher, J r.

Commissioner of Patenti Attesting Officer 

